*NCBI Gene # or RefSeq#: 15827075 *Protein ID (NP or XP #) or Wolbachia#:NP_301338.1 *Organism (including strain): Mycobacterium leprae strain TN Etiologic Risk Group (see link below): NIH RG 2 */Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium leprae causes an infectious disease called Leprosy (or commonly known as the Hansen's Disease). Leprosy affects the peripheral nerves, the skin, the upper respiratory tract, eyes, and the nasal mucus. Symptoms of Leprosy may show years later. Leprosy survive best at low temperatures and effects of this develops irrationally. Those with poor T-cell immunity towards M. leprae typically exhibit lepromatous (LL) leprosy or multi-bacillary leprosy, which is associated with symmetric skin lesions, nodules, plaques, thickened dermis, and frequent involvement of the nasal mucosa resulting in congestion and nose bleeds. Leprosy may cause some degree of peripheral neurological damage as well as muscle weakness. People with long-term leprosy may lose the use of their hands or feet due to repeated traumatic injury resulting from lack of sensation. If left untreated, it can cause progressive and permanent damage to the skin, nerves, eyes, and limbs.
Link to TDR Targets page (if present): click! Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: Serine-threonine protein kinase PknG is known to promote mycobacterial survival in macrophage. It does so by being secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake.
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer Complex of proteins?: monomer Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): corynebactieral PknG (CgPknG) - homology (click link!)
*EC#: 2.7.11.1
Link to BRENDA EC# page:click! -- Show screenshot of BRENDA enzyme mechanism schematic
Figure 1: Reaction catalyzed by serine/threonine protein kinase (2.7.11.1) from BRENDA.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) ---or link (or citation) to paper that contains assay information **Kinase assays** -- links to assay reagents (substrates) pages. See paper --- List cost and quantity of substrate reagents, supplier, and catalog # http://www.sigmaaldrich.com/catalog/product/sigma/cs0730?lang=en®ion=US Been discontinued- CS0730 but costs $766.00
Structure (PDB or Homology model)
Figure 2. PDB Crystal Structure of Protein Kinase PknG (4Y0X)
-- PDB # or closest PDB entry if using homology model: 4Y0X Current Inhibitors:K-252a, staurosporine, KbpA Expression Information (has it been expressed in bacterial cells): E. coli BL21 (DE3) Purification Method: His-tag Ni-NTA affinity purification
Image of protein (PyMol with features delineated and shown separately):
Figure 3. Pymol Structure of Protein Kinase PknG(4Y0X)shown as sticks. Carbons are colored as cyan and surface transparency set to 80%
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*GC% Content for gene:8.452536% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to Primer design results for 'tail' primers (this is just 2 sequences):
*Target (protein/gene name): Serine-threonine protein kinase PknG (protein kinase G) (STPK G)
*NCBI Gene # or RefSeq#: 15827075*Protein ID (NP or XP #) or Wolbachia#: NP_301338.1
*Organism (including strain): Mycobacterium leprae strain TN
Etiologic Risk Group (see link below): NIH RG 2
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium leprae causes an infectious disease called Leprosy (or commonly known as the Hansen's Disease). Leprosy affects the peripheral nerves, the skin, the upper respiratory tract, eyes, and the nasal mucus. Symptoms of Leprosy may show years later. Leprosy survive best at low temperatures and effects of this develops irrationally. Those with poor T-cell immunity towards M. leprae typically exhibit lepromatous (LL) leprosy or multi-bacillary leprosy, which is associated with symmetric skin lesions, nodules, plaques, thickened dermis, and frequent involvement of the nasal mucosa resulting in congestion and nose bleeds. Leprosy may cause some degree of peripheral neurological damage as well as muscle weakness. People with long-term leprosy may lose the use of their hands or feet due to repeated traumatic injury resulting from lack of sensation. If left untreated, it can cause progressive and permanent damage to the skin, nerves, eyes, and limbs.
Link to TDR Targets page (if present): click!
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein:
Serine-threonine protein kinase PknG is known to promote mycobacterial survival in macrophage. It does so by being secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer
Complex of proteins?: monomer
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): corynebactieral PknG (CgPknG) - homology (click link!)
*EC#: 2.7.11.1
Link to BRENDA EC# page: click!
-- Show screenshot of BRENDA enzyme mechanism schematic
Figure 1: Reaction catalyzed by serine/threonine protein kinase (2.7.11.1) from BRENDA.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
---or link (or citation) to paper that contains assay information **Kinase assays**
-- links to assay reagents (substrates) pages. See paper
--- List cost and quantity of substrate reagents, supplier, and catalog #
http://www.sigmaaldrich.com/catalog/product/sigma/cs0730?lang=en®ion=US
Been discontinued- CS0730 but costs $766.00
Structure (PDB or Homology model)
Figure 2. PDB Crystal Structure of Protein Kinase PknG (4Y0X)
-- PDB # or closest PDB entry if using homology model: 4Y0X
Current Inhibitors: K-252a, staurosporine, KbpA
Expression Information (has it been expressed in bacterial cells): E. coli BL21 (DE3)
Purification Method: His-tag Ni-NTA affinity purification
Image of protein (PyMol with features delineated and shown separately):
Figure 3. Pymol Structure of Protein Kinase PknG(4Y0X)shown as sticks. Carbons are colored as cyan and surface transparency set to 80%
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAESMKREHMDHDTEDVGQAAQRADPPSGTTEGRLQSTQAIFRPNFDDDDDLLHISVPSVDTEPQDRITP
ATRVLPPIRQLGGGLVEIRRVRDIDPLEALMTNPVVPESKRFCWNCGRPVGRSELEGQEADGAQGAKEGW
CPYCGSPYSFLPQLSPGDIVAGQYEVKGCIAHGGLGWVYLAFDHNVNDRPVVLKGLVHSGDAEAQASAVA
ERQFLAEVVHPQIVQIFNFVEHKDTSGDPVGYIVMEYIGGRSLKRGSKKGNVEKLPVAEAIAYLLEILPA
LSYLHSIGLVYNDLKPENIMLTEEQLKLIDLGAVSRINSFGCIYGTPGYQAPEIVRTGPTVATDIYTVGR
TLAALTLNLRTRNGRYMDGLPEDDPVLTTYDSFARLLHRAINPDPRRRFSSAEEMSAQLMGVLREVVAQD
TGVPRAGLSTIFSPSRSTFGVDLLVAHTDVYLDGRLHSEKLTAKDIVTALQVPLVDPTDVAAPVLQATVL
SQPVQTLDSLRAARHGMLDAQGIDLAESVELPLMEVRALLDLGDVVKANRKLDDLADRVSCQWRLVWYRA
VADLLTGDYASATKHFTEVLNTFPGELAPKLALAATAELAGESDEHKFYRTVWHTNDGVVSAAFGLARFQ
SAEGDRTGAVCTLDEVPPTSRHFTTARLTSAVTLLSGRSTNEITEQQIRDAARRVETLPPTEPRVLQIRA
LVLGCAMDWLADNQASANHILGFPFTKHGLRLGVEASLRSLARVAPTQRHRYTLVDMANKVRPTSTL
*length of your protein in Amino Acids 787 amino acidsMolecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 83888.2 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 67310
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4. TMpred output from the amino acid sequence of Protein Kinase PknG(4Y0X).*CDS Gene Sequence (paste as text only):
1 maesmkrehm dhdtedvgqa aqradppsgt tegrlqstqa ifrpnfdddd dllhisvpsv
61 dtepqdritp atrvlppirq lggglveirr vrdidpleal mtnpvvpesk rfcwncgrpv
121 grselegqea dgaqgakegw cpycgspysf lpqlspgdiv agqyevkgci ahgglgwvyl
181 afdhnvndrp vvlkglvhsg daeaqasava erqflaevvh pqivqifnfv ehkdtsgdpv
241 gyivmeyigg rslkrgskkg nveklpvaea iaylleilpa lsylhsiglv yndlkpenim
301 lteeqlklid lgavsrinsf gciygtpgyq apeivrtgpt vatdiytvgr tlaaltlnlr
361 trngrymdgl peddpvltty dsfarllhra inpdprrrfs saeemsaqlm gvlrevvaqd
421 tgvpraglst ifspsrstfg vdllvahtdv yldgrlhsek ltakdivtal qvplvdptdv
481 aapvlqatvl sqpvqtldsl raarhgmlda qgidlaesve lplmevrall dlgdvvkanr
541 klddladrvs cqwrlvwyra vadlltgdya satkhftevl ntfpgelapk lalaataela
601 gesdehkfyr tvwhtndgvv saafglarfq saegdrtgav ctldevppts rhfttarlts
661 avtllsgrst neiteqqird aarrvetlpp teprvlqira lvlgcamdwl adnqasanhi
721 lgfpftkhgl rlgveaslrs larvaptqrh rytlvdmank vrptstl
*GC% Content for gene: 8.452536%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to
Primer design results for 'tail' primers (this is just 2 sequences):